Cytogenetic Studies of B-cell Chronic Lymphocytic Leukemia (B-CLL)

B 细胞慢性淋巴细胞白血病 (B-CLL) 的细胞遗传学研究

• 批准号: 8554041
• 负责人: diane c arthur
• 金额: $ 10.3万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554041
• 关键词: 11q; 13q; 14q32; 17p; Abnormal Cell; Address; B-Lymphocytes; Blood Cells; Chromosomal Loss; Chromosome abnormality; Chronic Lymphocytic Leukemia; Clinical; Correlative Study; Cytogenetics; DNA; DNA Sequence Rearrangement; Data; Detection; Diagnosis; Disease; Disease Outcome; Evaluation; Fluorescent in Situ Hybridization; Frequencies; G-Banding; Gene Expression; In Vitro; Interphase; Karyotype; Laboratories; Leukemic Cell; Lipopolysaccharides; Metaphase; Microarray Analysis; Mitogens; Mitotic; Molecular Cytogenetics; National Heart, Lung, and Blood Institute; Outcome; Patients; Prospective Studies; Quality Control; Reporting; Risk; Series; Specificity; Techniques; Technology; Testing; Time; Treatment Protocols; Trisomy 12; Variant; base; cDNA Arrays; comparative genomic hybridization; disease characteristic; prognostic; response

Previous G-banded karyotype analyses detected clonal chromosome abnormalities in 40-100 percent of B-CLL patients studied after appropriate in-vitro stimulation with polyclonal B-cell mitogens. It was unknown whether the wide variations in frequencies of abnormalities reported in different series were due to true differences in disease characteristics or merely to in-vitro culture conditions. Common recurring chromosomal abnormalities included trisomy 12, rearrangements of 14q32, translocations or deletions of 13q, deletions of 6q, and deletions of 11q. Recent studies have shown increased detection of trisomy 12, 13q deletions, 11q deletions, and 17p (p53) deletions with interphase fluorescence in-situ hybridization (FISH) techniques, suggesting these changes do indeed occur early in the course of the disease. Data regarding the clinical and prognostic significance of cytogenetics in B-CLL are emerging. The data suggest certain karyotypic and/or FISH abnormalities are associated with specific clinicopathologic subsets of B-CLL, and with the course of the disease.To address the questions of timing of karyotypic changes in B-CLL, and the possible specificity and significance of these changes, we have been conducting prospective studies of patients with sporadic or familial B-CLL referred to the NCI and NHLBI for evaluation and possible treatment. The cytogenetics specific aims of this project are: to determine the optimal culture conditions for obtaining karyotypically abnormal mitotic cells from peripheral blood of patients with B-CLL; to determine whether or not interphase FISH detects clonally abnormal cells missed by G-banded metaphase analysis; to determine whether or not comparative genomic hybridization (CGH) will detect gains or losses of chromosomal material not found by metaphase or FISH analyses; and to correlate cytogenetics results with clinical, morphologic and immunophenotypic features of the disease, with cDNA microarray analysis of gene expression, and with outcome with new therapies. More than 250 patients have been entered on-study to date. Our initial analyses from 1997-2004 revealed clonal chromosome abnormalities in only 30-40 percent of cases using G-banded metaphase analysis, and demonstrated inferiority of e. coli lipopolysaccharide as a mitogen for the leukemic cells. Interphase FISH with a panel of five probes has confirmed or expanded the G-band findings in patients with abnormal clones, and has detected abnormalities in all but approximately 10% of patients tested to date. From 2002 through 2005 we performed CGH retrospectively on DNA isolated from patients previously entered on-study, and prospectively on DNA from new patients. CGH was less sensitive than interphase FISH in detecting submicroscopic deletions, but detected gains and losses of regions not probed by FISH. Combining G-banding, FISH, and CGH, we were able to find molecular cytogenetic abnormalities in more than 90% of B-CLL patients; FISH was clearly superior. Furthermore, dual hybridization with two probes and also sequential analyses in multiple patients have shown certain abnormalities present at diagnosis, and additional abnormalities at the time of transformation. Based upon these studies, we are now routinely performing interphase FISH on all patients. We are also quality-controlling new probes for addition to our routine FISH panel. Full G-banded karyotype analysis is done only if specifically indicated. Interphase FISH results are currently being used to classify patients with regard to risk, and in assignment to treatment protocols. Correlative studies of cytogenetics with clinical and other laboratory features at diagnosis and transformation, and with gene expression and response to treatment, are ongoing.

先前的g带核型分析发现，在适当的体外刺激多克隆b细胞有丝分裂原后，40- 100%的B-CLL患者中存在克隆性染色体异常。目前尚不清楚不同系列报告的异常频率的广泛差异是由于疾病特征的真正差异还是仅仅是体外培养条件的差异。常见的复发性染色体异常包括12三体、14q32重排、13q易位或缺失、6q缺失和11q缺失。最近的研究表明，使用间期荧光原位杂交（FISH）技术检测到12三体、13q缺失、11q缺失和17p （p53）缺失的增加，表明这些变化确实发生在疾病的早期。关于细胞遗传学在B-CLL中的临床和预后意义的数据正在出现。这些数据表明，某些核型和/或FISH异常与B-CLL的特定临床病理亚群和病程有关。为了解决B-CLL核型变化的时间问题，以及这些变化可能的特异性和意义，我们一直在对散发或家族性B-CLL患者进行前瞻性研究，并将其提交NCI和NHLBI进行评估和可能的治疗。本项目细胞遗传学的具体目的是：确定从B-CLL患者外周血中获得核型异常有丝分裂细胞的最佳培养条件；确定间期FISH是否检测到g带中期分析遗漏的克隆异常细胞；确定比较基因组杂交（CGH）是否能检测到中期或FISH分析未发现的染色体物质的增加或减少；并将细胞遗传学结果与疾病的临床、形态学和免疫表型特征、基因表达的cDNA微阵列分析以及新疗法的结果联系起来。迄今为止，已有250多名患者进入了研究阶段。我们从1997-2004年的初步分析显示，使用g带中期分析，只有30- 40%的病例出现克隆性染色体异常，并证明大肠杆菌脂多糖作为白血病细胞的有丝分裂原的劣性。由5个探针组成的间期FISH证实或扩大了异常克隆患者的g带发现，迄今为止，除了约10%的检测患者外，所有患者都检测到异常。从2002年到2005年，我们对先前进入研究的患者分离的DNA进行了回顾性的CGH，并对新患者的DNA进行了前瞻性的CGH。CGH检测亚显微缺失的灵敏度低于间期FISH，但检测到FISH未探测到的区域的增益和损失。结合g带、FISH和CGH，我们能够在超过90%的B-CLL患者中发现分子细胞遗传学异常；FISH显然更优越。此外，在多个患者中使用两个探针进行双杂交和序列分析显示诊断时存在某些异常，并且在转化时存在额外的异常。基于这些研究，我们现在对所有患者常规进行间期FISH。除了常规FISH面板外，我们还对新探针进行质量控制。完整的g带核型分析只在特殊情况下进行。间期FISH结果目前用于对患者进行风险分类，并分配治疗方案。细胞遗传学与诊断和转化的临床和其他实验室特征，以及与基因表达和治疗反应的相关性研究正在进行中。